Skeletal muscle has a unique capacity for repair after injury. This regenerative response fails in volumetric muscle loss injuries (VML), where a large volume of the muscle is damaged or removed, usually due to acute trauma. Current treatments for VML injury are limited, and often result in scar tissue formation and limited muscle function. Interest has been shown in developing myogenic scaffolds for VML repair. Clinical studies have investigated the use of decellularized extracellular matrix (dECM) laminated sheets derived from porcine bladder or small intestine submucosa (SIS) as acellular scaffolds for VML repair. ECM contains vital biologic factors (i.e. growth factors, basement membrane proteins, cryptic peptides) thought to be involved in the recruitment of progenitor cells, regulation of macrophage polarization, and tissue regeneration. VML injured muscles in animals and in humans implanted with dECM scaffolds have shown some muscle regeneration, including neovascularization and reinnervation in the scaffold, although the capacity of these muscles to generate force is still diminished compared to uninjured controls. Skeletal muscle exemplifies the structure- function relationship in biology; the capacity of a muscle to generate isometric force is directly related to the arrangement of fibers within a muscle. Histological examination of muscle in dECM scaffolds indicate poor fiber alignment with native muscle orientation, likely due to the lack of organized microstructure in the original dECM scaffolds, which is difficult to control using current fabrication techniques. We have developed a novel microscale continuous optical bioprinting (?COB) platform, which can be used to rapidly fabricate scaffolds with tissue informed microstructure and natural biomaterials (e.g. dECM) in 3D in a matter of seconds, providing a significant time and resolution advantage over traditional extrusion-based 3D printers. We broadly hypothesize that a scaffold consisting of dECM and an elastic, biocompatible material (acrylated poly (glycerol sebacate); PGSA) can promote organized muscle regeneration in a rat model of VML. In Aim 1, we propose to synthesize and fine-tune the formulation of PGSA, combined with dECM, to create an elastic, myogenic scaffold, with muscle informed microstructure using ?COB. In Aim 2, we will evaluate the capacity of the PGSA+dECM scaffold to regenerate skeletal muscle in a rat model of VML compared to tissue engineering solutions being explored in the clinic (laminated dECM sheets) at acute (2 weeks) and sub-acute (4 weeks) time points. 3D printing in healthcare represents the pinnacle for patient centric rehabilitation. This platform allows for physicians to design implants with any geometry, which can be directly extracted from standard presurgical imaging, to customize treatment for a patient. Future studies will evaluate the efficacy of this scaffold in larger animal models over longer time periods, with the long-term goal of clinical translation.